[Autoradiographic investigations on protein metabolism during limb regeneration in urodeles]

Abstract

Protein synthesis during limb regeneration has been studied by investigating the incorporation of thioamino acids-S-35 and leucine-H-3 in young postmetamorphicTriturus vulgaris andTr. alpestris. Amputation causes in all stump tissues considerable alterations of the amino acid incorporation rates. In epidermis, musculature and nerve sheaths incorporation is nearly doubled during the first hours after amputation. The increase of incorporation in proximo-distal direction is gradual.The protein synthesis of the epidermal cells decreases during wound closure when the cells are losing the connection with the stump epidermis. The reduced amino acid incorporation occurs with cytological alterations comparable to the typical aging processes of normal epidermal cells. During blastema multiplication and differentiation the protein synthesis of the epidermis reaches a maximum at about three times the rate of the proximal epidermis. Near the end of the differentiation phase this high rate of synthesis is reduced to 1.5 times of that of the normal epidermis.In the stump musculature a maximum of synthesis at the beginning of sarcolysis is shifted proximally and corresponds with the loss of the striation. In sarcoplasmic fragments without nuclei the progression in lysis is correlated to the breakdown of protein synthesis. In contrast sarcoplasmic fragments with nuclei intensify their protein synthesis. At the begin of blastema formation and multiplication their rate of synthesis reaches 10-15 times the amount of that of uninfluenced musculature. After amputation the incorporation rate of the sheath cells in the distal part of the nerves is doubled during the first days and increases furtheron. It reaches five times the rate of the proximal sheath cells.During blastema formation and multiplication the amino acid incorporation rate of the mesodermal blastema cells migrating distally increases steadily. Their highest incorporation rate has been found immediately before redifferentiation takes place. It is more than 30 times the rate of uninfluenced muscles in the proximal part of the stump and three times the rate of the proximal epidermis. The periosteum of the bone stump seems to react later than epidermis, muscles and nerves. Normally no extended lysis takes place. During the blastema formation stage the amino acid incorporation of the periosteum cells increases to five times the rate of that of the uninfluenced periosteum cells. At the begin of the redifferentiation of the regenerate it corresponds nearly to the rate of the mesodermal blastema cells. Together with the remaining fibrocytes in the stump they represent the blastematas to restitute the humerus and the connective tissue derivatives in the stylopodium. As soon as the specific determination of mesodermal blastema cells becomes visible protein synthesis is reduced. During histological differentiation synthesis increases again. The incorporation rates during histogenesis of the different tissues in the zeugopodium and autopodium are egual to the rates in corresponding tissues of the restituting stylopodium. The area of the presumptive dermis in the limb regenerate is characterized by cells with less protein synthesis than that of the mesodermal blastema cells.The investigations demonstrate that in each regeneration state protein synthesis in the different tissues and cell types occur with characteristic maxima, which often overlap eachother.